EAGER: Collaborative Research: Tracking of KOR1 Protein Transport in Arabidopsis using Fluorescent-Timer Imaging System

在这个项目中，将开发方法，允许直接观察蛋白质KORIGAN 1（KOR 1）在植物细胞中的运动。 KOR 1在纤维素纤维的形成中起作用，纤维素纤维是植物细胞壁的主要成分。由KOR 1控制的纤维素纤维沉积影响细胞生长和生物质生产。 KOR 1在细胞内从一个隔室移动到另一个隔室，然后最终到达细胞表面，纤维素纤维最终沉积在那里。 为了可视化KOR 1在植物细胞中的动态，该项目将应用荧光计时器（FT）技术，其中每个KOR 1蛋白质都标记有时间敏感的荧光标签。 由于FT随时间改变其颜色，因此表达FT标记的KOR 1的细胞的荧光图像将允许观察细胞内KOR 1的年龄和位置。 这将揭示KOR 1通过植物细胞的运输路线。 通过观察KOR 1转运在不同生物环境下的变化，这些研究将揭示某些因素如何影响蛋白质在细胞内的路径。 在植物中，KOR 1在不同的亚细胞结构域，如高尔基体网络（trans-Golgi network，TGN）、质膜（plasmamembrane，PM）、液泡膜（tonoplast，TP）以及细胞板（如分裂细胞）中的分布都得到严格的维持。KOR 1的功能障碍通常与TP靶向增加有关。 该项目的目的是可视化KOR 1运输的动力学，超越终点位置的分析。 将研究与KOR 1生物学相关的两个运输过程。 首先，将通过跟踪诱导蛋白质表达后KOR 1的年龄来分析从头合成的KOR 1的转运途径/动力学和重定向机制。 接下来，将使用KOR 1年龄信息来剖析细胞分裂期间KOR 1到细胞板的多个靶向途径。 这些系统将作为模型，进一步解剖定量影响KOR 1运输的各种因素。 该跨学科团队将优化FT技术用于植物细胞生物学，作为一个完整的荧光分析包，包括具有植物成熟时间特征的FT，兼容的荧光亚细胞标记物和自动化图像处理管道。本项目建立的实验平台将作为进一步研究影响KOR 1转运的多种因素的基础，包括蛋白质N-糖基化途径以及与纤维素合酶复合体的相互作用。

In this project, methods will be developed to allow direct observation of the movement of a protein, KORRIGAN1 (KOR1), in plant cells. KOR1 functions in the formation of cellulose fibers, which are the primary constituents of plant cell walls. Cellulose fiber deposition that is controlled by KOR1 influences cell growth and biomass production. KOR1 travels from one compartment to another inside cells before it finally reaches the cell surface where cellulose fibers will ultimately be deposited. To visualize the dynamics of KOR1 in plant cells, this project will apply fluorescent-timer (FT) technology, in which each KOR1 protein is labeled with a time-sensitive fluorescent tag. Because FT changes its color over time, fluorescence images of cells expressing FT-tagged KOR1 will allow visualization of the age and location of KOR1 inside of the cells. This will reveal the route of KOR1 transport through the plant cell. By observing how KOR1 transport changes under different biological circumstances, these studies will reveal how certain factors influence the paths that proteins travel within cells. This is a novel research tool/approach in plant biology that can change the way the scientists view proteins in any plant cell.In plants, distribution of KOR1 among different subcellular domains, such as trans-Golgi network (TGN), plasmamembrane (PM), tonoplast (TP), as well as the cell plate in case of the dividing cells, is scrupulously maintained. Dysfunction of KOR1 is often associated with increased targeting to TP. This project aims to visualize the dynamics of KOR1 transport beyond analysis of endpoint locations. Two transport processes relevant to KOR1 biology will be studied. First, transport route/kinetics and a redirection mechanism of de-novo-synthesized KOR1 will be analyzed by tracking the age of KOR1 after induction of protein expression. Next, KOR1 age information will be used to dissect the multiple targeting routes of KOR1 to the cell plate during cell division. These systems will serve as models for further dissection of various factors that quantitatively affect KOR1 transport. The interdisciplinary team will optimize FT technology for plant cell biology as a whole fluorescence analysis package, including FT with characterized in planta maturation time, compatible fluorescent subcellular markers, and automated image-processing pipelines. The experimental platform established in this project will serve as the basis for further investigation of multiple factors that affect KOR1 transport, including protein N-glycosylation pathways and interactions with cellulose synthase complex.